PROJECT SUMMARY/ABSTRACT 3q26 copy number gains (CNGs) have been found to be present in both early stage pre-invasive lung squamous cell lesions and in invasive lung squamous cell carcinomas (LSCCs). In addition, amplification involving the 3q26 region is consistently observed in high-grade, and less frequently in low-grade pre-invasive lesions. Furthermore, the presence of 3q26 amplification has been shown to accurately predict patients with endobronchial squamous metaplastic and dysplastic lesions that subsequently progressed to lung cancer. However, whether 3q26 CNG is an early oncogenic event that drives pre-neoplastic lesions to progress to malignant LSCC has not been adequately explored. Our studies have demonstrated that at least 3 genes (PRKCI, ECT2 and SOX2) on 3q26 are co-amplified and functionally collaborate in maintaining the transformed phenotype of LSCC cells. Our preliminary data demonstrates that 1) loss of Trp53 and overexpression of Sox2 induces expansion of mouse primary lung basal stem cells (LBSCs) ex vivo and formation of tumors that harbor squamous cell lineage markers in vivo; 2) forced co-overexpression of PKC?, Ect2 and Sox2 in Trp53 deficient mouse LBSCs further enhances expansion and causes morphological transformation ex vivo; and 3) Trp53 deficient LBSCs co-overexpressing PKC?, Ect2 and Sox2 exhibit PKC? mediated T328-Ect2 and T118-Sox2 phosphorylation. Based on these data, we hypothesize that PKC?, Ect2 and Sox2 are key oncogenic drivers of the 3q26 amplicon and together co-operate to mediate 3q26 CNG- driven LSCC tumorigenesis. These hypotheses will be tested by completing two interrelated specific aims designed to: 1) determine if PKC?, Ect2 and Sox2 co-operate in Trp53 deficient LBSC tumor formation in vivo and 2) assess the role of PKC?-mediated Ect2 and Sox2 phosphorylation and downstream signaling in Trp53 deficient LBSC morphological transformation ex vivo and tumor initiation in vivo. Successful completion of these aims will provide significant new insight into the role of multi-genic 3q26 amplification in LSCC development and progression.